鉴别猪伪狂犬病病毒强毒与疫苗毒双重PCR检测方法的建立

Development of Duplex PCR Method for the Differentiation of Virulent and Vaccine Strains of Pseudorabies Virus

根据猪伪狂犬病病毒(PRV)gE、gH基因序列,设计合成了2对特异性引物,分别建立gE和gH基因的单项PCR扩增方法。通过对PCR扩增条件的优化,建立了鉴别PRV强毒和疫苗毒的双重PCR检测方法,即利用一次PCR反应,从强毒株基因组中可同时扩增出2条大小分别为429 bp(gE基因)、355 bp(gH基因)的特异性片段,从弱毒株DNA中仅扩增出1条大小为355 bp的片段,而猪圆环病毒2型和猪细小病毒DNA扩增结果均为阴性。敏感性试验表明,所建立的双重PCR检测方法最低有效检测量为1×102TCID50/0.1 mL。该方法适合对PRV强毒和疫苗毒的快速鉴别诊断。

Based on the gE and gH genes of pseudorabies virus（ PRV）,two pairs of special primers were designed,respectively,single PCR assays for gE and gH genes were established. Under the optimized amplification conditions,the duplex PCR method was established to differentiate virulent and vaccine strains of pseudorabies virus. In a duplex PCR assay,two fragments of 429 bp for gE gene and 355 bp for gH gene were simultaneously amplified from pseudorabies virus virulent strains DNA,only a 355 bp fragment for gH gene was amplified from pseudorabies virus vaccine strains DNA,whereas no PCR products were amplified from porcine circovirus type 2 and porcine parvovirus. Then the detection limit of duplex PCR was estimated to be 1 × 10^2TCID50/0. 1 mL. This method is suitable for differentiation of wild-type pseudorabies virus and vaccine strains in clinical specimen.